It is known that the spatial resolution in measuring a sample in z-direction is increased in a confocal microscope by means of a pinhole diaphragm arranged at a point which is equivalent to the desired measuring point. In a microscope of the kind described at the beginning this increase is achieved with regard to all measuring points. I.e. the pinhole diaphragms of the pinhole diaphragm array are arranged in the beam path of the sample light at points which are equivalent to the measuring points. At the same time, the position of the measuring points is defined by the division of the ray bundle of illumination light into the partial ray bundles by means of the microlens array as each measuring point is the projection of a focus of a convergent partial ray bundle into the sample. In so far, the microlens array and its arrangement have to correspond to the pinhole diaphragm array and its arrangement.
A confocal scanning fluorescence microscope comprising two microlens arrays and a pinhole diaphragm array is known from U.S. Pat. No. 5,717,519 A. Here, the microlens array is realised as a microlens wheel, which is arranged in parallel to a so called Nipkow-disc which forms the pinhole diaphragm array. The microlens wheel and the Nipkow-disc may be rotated about a common rotating axis running perpendicular to their respective planes of main extension so that a scanning device for the sample is realized. A Nipkow-disc is a rotating disc with a spiral-shaped arrangement of pinhole diaphragms around the rotating axis. In the known confocal microscope the ray bundle of illumination light first passes through the microlenses of the microlens wheel. As a result, the illumination light is split up into a plurality of convergent partial ray bundles. The focus of each partial ray bundle is in the area of the passage way of a pinhole diaphragm of the Nipkow-disc. The beam splitter which deviates the sample light coming from the sample through the Nipkow-disc in front of the microlens wheel laterally towards a detector is arranged between the microlens wheel and the Nipkow-disc. Particularly, the beam splitter is a dichroitic mirror, which also results into an undesired deflection of the convergent partial ray beams coming from the microlens wheel. To compensate for this deflection, the microlens array and the Nipkow-disc in the known confocal microscope are tilted by a small angle towards the beam axis of the incident beam bundle of the illumination light coming from a laser. In the known confocal microscope the sample light does not pass through the microlenses of the microlens wheel; instead it is prior to that deflected by the dichroitic mirror laterally towards the detector. The high laborious adjustment of the microlens array with regard to the Nipkow-disc is a disadvantage of the known confocal microscope. If essentially the full illumination light coming from the laser is to be used for illuminating the sample in the measuring points, the Nipkow-disc has to be exactly orientated in such a way, that each focus of each convergent partial ray bundle coming from the microlens array exactly falls in the passage way of a pinhole diaphragm of the Nipkow-disc. This means high demands with regard to the parallelism of the microlens wheel and the Nipkow-disc, with regard to their distance and with regard to their rotational orientation about the common rotation axis. Further, the absolute orientation of this rotation axis has to be adjusted exactly to realize the desired compensation for the deflection of the partial ray bundles by the dichroitic mirror. In all that, it has to be considered that the beam splitter is arranged between the m microlens wheel and the Nipkow-disc and that the beam path of the sample light in radial direction from the common rotation axis of the microlens wheel and of the nipkow-disc should not even temporarily be interrupted.
A scanning fluorescence microscope is known from WO 98/28775 A in which the pinhole diaphragm array is omitted for avoiding the laborious adjustment of a pinhole diaphragm array with regard to a microlens array. The spatial resolution in z-direction of the known microscopy is realized by means of a simulation of a pinhole diaphragm array in the area of the detector by means of software, or by means of a two-photon-excitation of the sample in the measuring points. However, the effect of a real pinhole diaphragm array increasing the spatial resolution, i.e. the spatial resolution in z-direction of a real confocal arrangement can not be achieved by a simulating of a pinhole diaphragm array in the area of the detector, and the yield of sample light is comparatively low with a two-photon-excitation of a sample.
Thus, there is a need for a confocal microscope comprising two microlens arrays and a pinhole diaphragm array in which the actual adjustment labour is reduced, and which as a result can be realized at lower cost.